Membrane proteins and secreted proteins are expressed, folded, and glycosylated in eukaryotic cells as they pass along the secretory pathway from the endoplasmic reticulum (ER) through the Golgi apparatus to the plasma membrane in a process known as protein trafficking. There are now numerous examples of genetic diseases that arise when mutations in proteins result in improper folding, including familial hypercholesterolemia, α1-antitrypsin deficiency, congenital long QT syndrome, Fabry disease, and cystic fibrosis (Moyer, B. D. and Balch, W. E., Emerging Therapeutic Targets 5 165-176 (2001)). Misfolded proteins are recognized as defective by the cell's quality control mechanisms in the ER and are degraded. Because the misfolded protein fails to traffic to the Golgi apparatus and beyond, these diseases are commonly referred to as diseases of protein trafficking or protein misfolding.
Cystic fibrosis (CF) is an example of a disease of protein misfolding. CF is the most common lethal genetic disease in Caucasians, with approximately 60,000 affected individuals in Europe and North America. Cystic fibrosis is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein (Riordan, J. R., Ann. Rev. Physiol. 55 609-630 (1993)). CFTR is a plasma membrane protein which belongs to the ATP-binding cassette family of membrane transport proteins. It acts as a cyclic adenosine monophosphate (cAMP)-gated chloride channel as well as a regulator of other ion transport proteins (Welsh, M. J. et al., Ch. 201 Cystic Fibrosis, in The Metabolic and Molecular Bases of Inherited Disease, 8th Ed. online, Ed by Scriver, C. L. et al., McGraw Hill, 2001). While hundreds of different CFTR mutations are known, almost ⅔ of CF patients are homozygous for a mutation in which phenylalanine 508 is deleted (ΔF508). ΔF508 CFTR does not fold properly in the endoplasmic reticulum, and is degraded.
CF is a disease of the epithelial tissues, including the lungs, pancreas, sweat glands, and vas deferens. CFTR is expressed in the apical membrane of epithelial cells and the ΔF508 mutation causes a loss of chloride transport function at this site. In the airways, loss of CFTR function is thought to result in changes in the quantity or 10 composition of the airway surface liquid. Consequently, mucociliary clearance is hindered and the patient is predisposed to bacterial lung infections. Mortality is the result of a progressive loss of lung function arising from the collateral damage from the immune response to chronic infections (Rosenstein, B. R. and Zeitlin, P. L., Lancet 351 277-282 (1998)).
ΔF508 CFTR is known to retain chloride channel activity, albeit at a lower level than wild-type CFTR, and a small amount of ΔF508 CFTR is able to traffic to the plasma membrane. There have been attempts to correct the ΔF508 CFTR folding defect or compensate for its effects using pharmacologic agents. Small molecules such as glycerol, dimethylsulfoxide, deuterated water, and trimethylamine-N-oxide (TMAO) are known to promote protein folding at high concentrations (Brown, C. R. et al., Cell Stress and Chaperones 1 117-125 (1996)). These so-called ‘chemical chaperones’ are capable of restoring some chloride channel function in cells expressing ΔF508 CFTR, and TMAO has been shown to be effective in mice, but the high concentrations required make their use as therapeutics in man impractical (Fischer, H. et al., Am. J. Physiol. 281 L52-L57 (2001)).
Compounds that increase CFTR synthesis or decrease degradation have also been evaluated. The short-chain fatty acid butyrate is thought to increase CFTR protein synthesis to a level that overwhelms the quality control apparatus and allows additional mutant CFTR to reach the plasma membrane (Cheng, S. H. et al., Am. J. Physiol. 268 L615-L624 (1995)). An analog, phenylbutyrate (PBA), is thought to assist CFTR trafficking by modulating the levels of heat shock proteins involved in the folding of CFTR and its targeting for degradation. Phenylbutyrate is an approved drug used to treat urea cycle disorders and has been evaluated in CF patients. While high doses of PBA led to detectable response in nasal potential difference measurements (Zeitlin, P. L. et al., Molecular Therapy 6 119-126 (2002)), there has so far been no convincing demonstration of a robust therapeutic effect.
Deoxyspergualin, an inhibitor of Hsp70 chaperone function, has been shown to restore ΔF508 CFTR trafficking to some extent (Jiang, C. et al., Am. J. Physiol. 275 C171-C178 (1998)), but its broad immunosuppressive effects likely preclude its use as a drug. Inhibitors of the ER calcium pump, such as thapsigargin and dibutylhydroquinone, have been shown to increase the expression of functional ΔF508 CFTR in cells and in mice, presumably by releasing mutant CFTR from calcium-dependent chaperone proteins (Egan, M. E. et al., Nature Medicine 8 485-492 (2002)). These compounds are unlikely to become drugs because altering ER calcium levels will affect the trafficking of many other proteins in addition to CFTR.
Substituted benzo[c]quinolizinium compounds have been discovered that increase chloride transport activity in cells expressing ΔF508 CFTR, including cells from a CF patient (Dormer, R. L. et al., J. Cell Science 114 4073-4081 (2001)). These compounds are active at relatively high concentrations and to date, no toxicity or pharmacokinetic data and no efficacy data in CF patients has been reported.
Another strategy has been to attempt to increase the activity of the small amount of mutant CFTR present at the plasma membrane. Genistein, a naturally occurring isoflavone, has been found to increase the open probability of the CFTR channel (Hwang, T. -C. et al., Am. J. Physiol. 273 C988-C998 (1997)). Another compound, 8-cyclopentyl-1,3-dipropylxanthine (CPX), appears to increase open probability and also assist trafficking by binding directly to CFTR (Arispe, N. et al., J. Biol. Chem. 273 5727-5734 (1998)). Both compounds are currently being evaluated in clinical trials.
To date, none of these compounds have been shown to be effective in treating cystic fibrosis. Likewise, gene therapy to introduce wild-type CFTR into epithelial cells has failed to show therapeutic efficacy thus far. Consequently, there exists an unmet medical need for compounds that restore CFTR function by assisting protein folding and trafficking. Once such compounds have been identified it is likely that they will find broader use in other diseases of protein misfolding.